Method and device for antibiotic susceptibility testing based on fluctuations of electrical resistance in a microchannel

ABSTRACT

A system and method for antibiotic susceptibility testing efficiently determines whether bacteria are alive or have been killed by antibiotic treatment. The antibiotic susceptibility testing device includes at least one reservoir into which a bacteria solution is introduced and a microfluidic channel connected to the reservoir, wherein the cross-sectional size of the microfluidic channel is selected to be comparable to the size of the bacterium to be tested. Furthermore, the electrical resistance or voltage signal across the microchannel is monitored as bacteria swim into and out of the channel. Alternatively, a small population of bacteria can be immobilized in the microchannel. The resistance or voltage signal fluctuates when the bacteria are alive and moving in and out of the channel or wiggling on the microchannel walls. If the bacteria are dead, they have limited motility and the signal fluctuations are significantly smaller. By monitoring the signal fluctuations, the antibiotic susceptibility testing device can determine whether or not bacteria are alive, thus enabling antibiotic susceptibility testing of bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims any and all benefits as provided by lawincluding benefit under 35 U.S.C. §119(e) of the U.S. ProvisionalApplication No. 62/371,417, filed Aug. 5, 2016, the contents of whichare incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

TECHNICAL FIELD

The present invention relates to an antibiotic susceptibility testingapparatus for determining whether or not bacteria are live, and moreparticularly antibiotic susceptibility testing of bacteria.

BACKGROUND

Multi-drug resistant bacteria affect 2 million people in the U.S. everyyear, resulting in 23,000 deaths and causing an economic burden of anestimated $20 billion. The problem is exacerbated by the fact thatdevelopment of new antibacterial agents has slowed down in the pastdecade. In short, bacteria are gaining resistance to availableantibiotics at a rate faster than new antibiotics can be developed andbrought to market.

In order to impede the takeover by resistant strains and to preserve theexisting antibiotics, physicians must improve the process of prescribingantibiotics. Ideally, an antibiotic therapy should start only afterconfirming the susceptibility of the infecting bacteria to theantibiotic. However, physicians typically treat serious infectionsempirically by prescribing broad-spectrum antibiotics, because standardantibiotic susceptibility tests (ASTs) require long cell culturingsteps. An antibiotic susceptibility test (AST) determines whether or notbacterial isolates from a patient's blood, wound specimens, or urine aresusceptible to administered antibiotics. One of the gold standard tests,a broth dilution test, is performed by preparing a set of bacteriasolutions, which are incubated overnight in the presence of differentantibiotics with different concentrations. If the bacteria are resistantto the antibiotic, they multiply and the solution eventually becomesturbid. The limits of a typical optical measurement require the bacteriasolution to be incubated for 16-20 hours. Similarly, the disk diffusionmethod is performed by applying a bacterial inoculum to the surface ofan agar plate, on which antibiotic disks are placed. After incubationfor 16-24 hours, the antibiotic inhibits the bacteria growth in theregions where it diffuses. The susceptibility of the bacterial straincan be determined from the size of the bacteria-free zones around theantibiotic disks.

However, both these tests have a shared shortcoming: one has to waitlong enough such that the population reaches minimum detectable growthlevels. Accordingly, there exists in the art a need for novel, rapid,sensitive and robust methods to determine the antibiotic susceptibilityof bacteria.

It is clear that the above-described mainstay methods have limitations,and multi-drug resistant bacteria pose a grand challenge in globalhealthcare. Hence, the development of rapid antibiotic susceptibilitytests (ASTs) is an active research area, with many publications andpatents. The focus is on observing bacterial resistance at early stagesof cell growth. Polymerase chain reaction (PCR) is the quintessentialgenotypic method. PCR directly detects the resistance gene of a verysmall bacterial sample and can provide fast identification of antibioticresistance. However, it has limited utility, because only a fewresistance genes are firmly associated with phenotypic antibioticresistance. There are simply too many genetic mutations, acquisitions,and expressions to be routinely identified by current PCR techniques.Further, the use of PCR at point-of-care settings remains challenging.

Phenotypic methods, on the other hand, are based on observablecharacteristics of bacteria. Novel phenotypic ASTs typically employmicrofluidics and microdevices, because these devices allow foreffective use of samples and multiplexing. Researchers have beenexploring different approaches based on microfluidics. In the first typeof experiments, the growth of bacteria was directly observed in smallvolumes (e.g., microfluidic channels) in order to determinesusceptibility to different antibiotics. This approach was pushed downto the single cell limit by confining cells in drug-infused nano andpico-liter droplets. In another approach, bacteria were adhered tooscillating microstructures, such as microcantilevers or magneticmicrobeads, under administered antibiotics; here, the oscillationfrequency decreased due to added bacteria mass if the bacteriamultiplied, indicating resistance. Finally, measuring the physical orchemical properties of a medium (e.g., changes in electrical impedanceor pH) due to bacterial proliferation allowed for measuring microbialgrowth. The changes with administered antibiotics then provided thedesired susceptibility testing.

While most of the above-mentioned techniques are ingenious, very fewhave found their ways into the mainstream. There are still several majorchallenges. Some techniques require delicate microscopy or are toocomplicated to be implemented at point-of-care settings. Others relyupon labeling (e.g., fluorescent labeling), which limits utility. If thetest determines susceptibility based on whether or not the bacteria aregrowing, the heterogeneity of the antimicrobial response of bacteriabecomes an issue. In summary, there is a significant need for newapproaches.

SUMMARY

The present invention is directed to methods and system for antibioticsusceptibility testing. Specifically, the antibiotic susceptibilitytesting system according to the invention includes at least onereservoir into which a bacteria solution can be introduced. The testingsystem includes a microfluidic channel connected to the reservoir andthe bacteria in the solution are free to swim into the microfluidicchannel. The resistance or conductivity of electric currents through thechannel can be monitored as the bacteria swim into and out of themicrofluidic channel. The movement of the live bacteria into and out ofmicrofluidic channel can be detected as fluctuations in the measuredresistance or conductivity. A related approach is based oncapturing/immobilizing a small population of bacteria (˜50 cells) insidethe tiny microfluidic channel and the nanomechanical fluctuations ofsurface-adhered bacteria can be measured as fluctuations in the measuredresistance or conductivity. A signal measuring device can be used tomeasure changes in the current or voltage through the microchannel overtime. For example, the signal measuring device can include anoscilloscope, a digital signal processor, an analog signal processor, anammeter, a capacitance meter, a multimeter, a computer, a programmablecontroller, etc.

The measured signal fluctuations in the resistance (or conductivity)caused by live bacteria can be distinguished from signals produced bydead bacteria in solution because dead bacteria have limited motilityand the baseline fluctuations in the signal are significantly smaller(e.g., caused by thermal and Brownian motion) than the signalfluctuations when live bacteria are present. Accordingly, a method andsystem for testing antibiotic susceptibility is provided whereby abacterial solution can be introduced into the system and a baseline ofresistance (or conductivity) can be determined and used to evaluate anychange in resistance (or conductivity) after the introduction ofantibiotics into the reservoir to determine susceptibility. The systemcan determine the Root Mean Square (RMS) resistance, current or voltageof the signal fluctuations and compare the RMS signal value obtainedfrom the microchannel when the bacteria are in the microchannel to thebaseline RMS signal value obtained before the bacteria enter themicrochannel to determine the presence of live bacteria in themicrochannel.

This differs from Coulter counters, flow cytometers and other similardevices, wherein particles or cells are pushed through a constriction,either by a flow or by an electric field, and counted (i.e., detected)one by one.

The present invention provides a new paradigm of detection. The activityof live bacteria can be detected, monitored and characterized, forexample, for determining the viability of the bacteria. The activity ofdead bacteria can be distinguished because the signal fluctuations aresignificantly reduced—dead bacteria move only due to Brownian andthermal motion, which is smaller by orders of magnitude.

One aspect of the technology described herein relates to an antibioticsusceptibility testing system adapted to include a reservoir directlyconnected to a microchannel and bacteria from the reservoir are free tomove into the microchannel. The microchannel can further include asecond, antibiotic reservoir directly connected to the microchannel bypores that are large enough to pass the antibiotic but small enough toblock the bacteria. Once the antibiotic is introduced into theantibiotic reservoir it quickly diffuses into the microchannel region,and the susceptibility testing can begin. The fabrication of the porescan be achieved by using advanced lithography (e.g., electron beamlithography); alternatively, a porous material can be used as the topwall of the device, instead of polydimethylsiloxane (PDMS). In anexemplary embodiment, high currents (˜1 mA) can be implemented to drivethe bacteria in solution through the microchannel (e.g., through thechip).

In accordance with some embodiments of the invention, the bacteria canbe pushed into the microchannel region efficiently by using highcurrent/voltage. For example, this embodiment can be used to test in lowconcentration solutions, in which an applied high current can be used tospeed up the bacteria arrival into the microchannel. In accordance withsome embodiments of the invention, electrodes can be provided to form anelectrical trap to trap the bacteria in the microchannel for moreefficient detection. As a result, the antibiotic susceptibility testingsystem can be varied where electrical guiding/trapping electrodes aredesigned on another layer and integrated into the device.

In accordance with some embodiments of the invention, a solution ofnon-motile and motile bacteria can be introduced into the device from afirst reservoir. A voltage can be applied causing the bacteria to bepushed to a constriction at the end of the microchannel. After thebacteria have accumulated in the constriction, a low current can beapplied to measure the resistance (or voltage or current) signalfluctuations produced by presence of live bacteria in the channel.

In accordance with some embodiments of the invention, a solution ofnon-motile or motile bacteria can be introduced into the device from afirst reservoir. A pressure-driven flow can be created causing thebacteria to be pushed to a constriction at the end of the microchannel.After the bacteria have accumulated in the microchannel, a low currentcan be applied to measure the resistance (or voltage or current) signalfluctuations produced by presence of live bacteria in the channel.

In accordance with some embodiments of the invention, a solution ofnon-motile or motile bacteria can be introduced into the device from afirst reservoir. The microchannel can be coated with anadhesion-promoting surface layer. This will increase the efficiency ofbacteria accumulation. Alternatively, the microchannel can be coatedwith a layer of specific antibody selected to trap specific bacteriasuch that the selected bacteria accumulate inside the microchannel.After the bacteria have accumulated in the microchannel, a low currentcan be applied to measure the resistance (or voltage or current) signalfluctuations produced by presence of live bacteria in the channel.

In accordance with some embodiments of the invention, the geometry ofthe microfluidic channel can be varied to attain more efficientdetection of the bacterial movements and the signal fluctuations.

In accordance with some embodiments of the invention, the electricalmeasurements of the microfluidic channel may be varied to attain moreefficient detection of the bacterial movements signal fluctuations.

In accordance with some embodiments of the invention, motile andnon-motile bacteria can be immobilized on the channel walls; theirmechanical movements and fluctuations will still modulate the electricalconductance/resistance of the microchannel. This embodiment can be usedto evaluate bacteria such as staph infections.

In accordance with some embodiments of the invention, a direct current(e.g., a constant or near constant direct current) can be applied acrossthe microchannel and the bacteria movements can be monitored as afluctuating voltage signal or an AC voltage signal. Alternatively, a DCvoltage (e.g., a constant or near constant DC voltage) can be appliedacross the microchannel and the bacteria movements can be monitored aselectrical current signal fluctuations.

In accordance with some embodiments of the invention, a correlationmeasurement can be performed. In this embodiment, a second pair ofelectrodes can be inserted but through a separate path. The current canbe applied as before through the first electrode pair. Then, the voltagefluctuations measured through electrode pair A is cross-correlated withthose measured through B: <V_(A)(t)V_(B)(t)>. This can be used toprovide a more sensitive measurement of the signal fluctuations and canbe used to avoid signal artifacts from outside the microchannel region(i.e., the bacteria).

In accordance with some embodiments of the invention, radiofrequency(RF) signal measurements can be performed. As a general matter, RFsignal measurements can provide a number of advantages overlow-frequency measurements. First, the availability of low-noise RFamplifiers can make RF measurements more precise. Second, RFmeasurements will provide much larger bandwidths (much smaller timeresolution). Furthermore, in these measurements, one does not need toapply a direct current through the nanochannel. Instead, one electrodecan be grounded and the other can be connected to a reflectometer. Thismeasurement can be used to determine (a complex measure of) theimpedance of the nanochannel as a function of time. The imaginary partof the impedance can change more strongly than the real part (i.e.,resistance), making this measurement useful for bacteria as well asother particles. Alternatively, RF signal transmission through themicrochannel can be measured. In accordance with some embodiments,impedance matching (e.g., an LC tank circuit) can be used to bettercouple the power to the microchannel region of the circuit.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing (s) will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A, 1B, and 1C shows an exemplary antibiotic susceptibilitytesting system in accordance with some embodiments of the invention.There are four mm-scale reservoirs connected to large channels; at thecenter is the tiny microchannel.

FIG. 2A exemplifies an ionic solution filling the microchannel, whereasFIGS. 2B and 2C show exemplary voltage fluctuations with thecorresponding activity (e.g., movements) of bacteria in accordance withsome embodiments of the invention. FIG. 2D shows exemplary voltagefluctuations with the bacteria trapped or captured to the microchannelwalls in accordance with some embodiments of the invention. In FIG. 2D,the physical or chemical bond between the bacterium and the surface ismodeled as a spring in accordance with some embodiments of theinvention. FIG. 2E shows exemplary mean resistance increase (under aconstant applied current) as the bacteria proliferate in accordance withsome embodiments of the invention. FIG. 2F exemplifies a chip designedwith several multiplexed microchannels connected to a reservoir toenable comparison to the measured electrical signals in the microchannelbefore and after administering the antibiotic in accordance with anembodiment of the invention.

FIGS. 3A-3C exemplify testing performed to demonstrate the efficiency ofthe antibiotic susceptibility testing system in accordance with someembodiments of the invention.

FIG. 4 shows an example of a workflow for antibiotic susceptibilitytesting including electrical fluctuation based susceptibility testingand mean resistance based susceptibility testing, in accordance withsome embodiments of the invention.

FIGS. 5A-5C show variations of the antibiotic susceptibility testingsystem in accordance with some embodiments of the invention. FIG. 5Ashows an embodiment that demonstrates the addition of an added reservoirconnected to the microchannel directly. FIG. 5B shows electrodesdesigned and fabricated on the chip for controlling and trapping thebacteria in the microchannel region for faster and more efficientmeasurements in accordance with some embodiments of the invention. FIG.5C exemplifies another variation of the antibiotic susceptibilitytesting system for non-motile bacteria in accordance with someembodiments of the invention.

FIGS. 6A-6E show examples of a process for bacteriacapture/immobilization in the microchannel. FIGS. 6A, 6B and 6C showbacteria are pipetted into the top reservoir; either a pressure gradientor an electric field pushes them into the microchannel where they arecaptured by surface chemistry, jammed into the constriction by the flow,or simply stick to the microchannel surface on their own. FIG. 6D showsapproximately 50 E. coli immobilized in a tapered microchannel. Theentire taper region has a height of ˜1 μm, and thus confines thebacteria. Once immobilized, the bacteria do not leave the shown taperregion but continue their nanomechanical movements (wiggling). The insetshows the electrostatic interaction mediated by an adhesion-promotingcoating, PDL. This non-specific chemistry increases the captureefficiency, but it is not essential. Alternatively, one could use aspecific surface coating (e.g., antibody) to selectively capturebacteria in the microchannel in a targeted manner. FIG. 6E showsenhancement of local density by capture in the microchannel wherebacteria (S. epidermidis) are introduced into the device at t=0, and aflow is established by a pressure gradient. The initial concentration ofthe bacteria in solution is about 10⁵ colony forming units (CFU)/ml. Thevolume of the V-shaped (tapered) microchannel region is about 10⁻⁸ ml.After 2 minutes, the flow deposits about 10 bacteria in themicrochannel, resulting in a local density of about 10⁹ bacteria/ml.After 5 minutes, the local density goes up by another factor of two toabout 2×10⁹ bacteria/ml. (Bacteria are highlighted in red.)

FIGS. 7A and 7B show that the bacteria can be adhered or otherwiseattached to the surface of the walls of the microchannel and can bemodeled as a bacteria attached to the wall by a spring.

FIG. 8A shows an electrostatic interaction between the channel wall,PDL, and bacteria in order to adhere bacteria on the microchannel wallsin accordance with some embodiments of the invention. FIG. 8B shows thenanomechanical models of the bacteria adhered on the microchannel walls.The springs model the PDL and are electrostatic in nature, in accordancewith some embodiments of the invention.

FIG. 9A shows another pair of electrodes inserted through separate pathsin accordance with some alternative embodiments of the invention. FIG.9B exemplifies implementing a basic RF reflectometry measurement inaccordance with some alternative embodiments of the invention. FIG. 9Cexemplifies implementing an alternative RF transmission measurement inaccordance with some alternative embodiments of the invention.

FIG. 10 shows an alternative antibiotic susceptibility testing systemwhere there exists a significant reduction of sample volume inaccordance with some embodiments of the invention.

FIG. 11 shows the detection of movements of surface immobilized motilebacteria, E. coli. wherein the red trace shows the time-dependentelectrical fluctuations due to nanomechanical movements of about 50cells (inset). The black data trace is the background signal before thebacteria are introduced into the microchannel.

FIGS. 12A and 12B show the electrical fluctuations caused by movementsof non-motile bacteria, S. epidermidis, wherein the cells are capturedin a tapered microchannel (inset). FIG. 12A shows the electricalfluctuations in the time domain before (black baseline signal) and after(red) the cells are captured. FIG. 12B shows the power spectra of thesignals shown in FIG. 12A. The nanomechanical movements of the bacteriacause increased electrical fluctuations and increase in RMS signal.

FIGS. 13A-13C show antibiotic susceptibility test results from bacterialmovements in human urine. FIGS. 13A and 13B show voltage fluctuations inone-minute long electrical measurements, over time, on a susceptible E.coli strain (FIG. 13A, in red) and a resistant E. coli strain (FIG. 13B,in blue). FIG. 13C shows a bar chart of the RMS magnitude of thefluctuations. The “before-antibiotic” amplitudes vary from sample tosample, because they depend on the size of the bacteria population andwhere the bacteria are trapped in the taper.

FIGS. 14-16 show viability detection and AST from proliferation in amicrochannel. FIG. 14 shows microscope images of the microchannel filledwith only the Bacteria (S. epidermidis) at t=0, 1 hr., 2 hrs., 3 hrs.and 4 hrs. FIG. 15 shows microscope images of the microchannel filledwith the Bacteria (S. epidermidis) and an effective antibiotic at t=0, 1hr., 2 hrs., 3 hrs. and 4 hrs. FIG. 16 shows a bar chart of the changein mean resistance that reflects the size of the bacteria population inbroth (blue bars on the right—no antibiotics) and in antibiotics/broth(green bars on the left) solutions. The microscope images shown in FIGS.14 and 15 are snapshots of the bacteria and show how the techniqueworks. The resistance changes shown in FIG. 16 are given with respect tothe empty microchannel resistance.

DETAILED DESCRIPTION

Aspects and embodiments of the invention relate to an antibioticsusceptibility testing system comprising at least one reservoir intowhich a bacteria solution is introduced and a microfluidic channel(herein referred to as a microchannel or a nanochannel) connected to thereservoir which supplies the bacteria solution for testing. Theantibiotic susceptibility testing system can be used to determinewhether or not bacteria are live and viable, thus efficiently enablingantibiotic susceptibility testing of bacteria.

The antibiotic susceptibility testing system according to the inventiondetects bacterial activity (e.g., natural motility, viability). By usingsmall devices (nano-scale devices), signals from a few bacteria can bedetected and thus enabling detection of a much smaller bacteriapopulation. This approach can significantly reduce the complexity andtime necessary for sample testing, and hence the sample-to-answer time.The antibiotic susceptibility testing system according to the inventionis based on a simple electrical (e.g., resistance or conductance)measurement. Hence, it does not require sensitive microscopy, is labelfree, and can be used at the clinical point of care.

FIGS. 1A, 1B and 1C show diagrams of an antibiotic susceptibilitytesting system 100 in accordance with some embodiments of the invention.The antibiotic susceptibility testing system 100 can comprise at leastone reservoir 110 coupled to at least one microchannel 120. As shown inFIG. 1, the system 100 can include four mm-scale reservoirs 110. Metalelectrodes for electrical measurements can be inserted into thereservoirs 110 or integrated in to them during fabrication. Themicrochannel 120 can be at the center of the system 100. Saline orbacteria solutions (e.g., an ionic solution such as urine, a buffersolution or other biological matrix) can be introduced by pipetting orpumping liquid into at least one of the reservoirs 110. The liquidsolution can be wicked (e.g., via capillary action) or pumped (e.g., viapressure) into the microchannel 120. FIG. 1A shows (inset) a microscopeimage of a polydimethylsiloxane (PDMS) device, showing the tinymicrochannel 120 in between reservoirs 110. The voltage and/or currentcan be measured by a signal measuring device V (e.g., an oscilloscope, adigital signal processor, an analog signal processor, an ammeter, avoltmeter, or a computer) and the fluctuation in voltage (or current)can be used to detect the presence of live bacteria. The signalmeasuring device V can be configured to store and display the amplitudeof the signals over time (e.g., a graph, chart, running average, peakdetection, etc.). The signal measuring device V can include a computeror microprocessor and associated memory that stores programs to beexecuted that can process the signal (e.g., the resistance, voltage,and/or current) to determine the presence of live bacteria in thechannel as a function of the electrical signal fluctuations measured inthe microchannel 120.

FIGS. 1B and 1C show diagrammatic views of an antibiotic susceptibilitytesting system 100 in accordance with some embodiments of the invention.FIG. 1B shows a diagram of a microfluidic transducer chip 101 inaccordance with some embodiments of the invention. FIG. 1C shows adiagram of a circuit model for the microfluidic transducer chip 101shown in FIG. 1B. The microfluid transducer chip 101 can be formed fromPDMS and bonded onto a glass substrate 102. In accordance with someembodiments of the invention, microfluidic transducer chip 101 caninclude four mm-scale reservoirs 110 connected to 100-μm-depth channels112. At the center, the tiny microchannel 120 connects the channels 112.Preferrably, the microchannel 120 has cross-sectional dimensionscomparable to the size of the bacteria in the range from 100nanometers—several micrometers (e.g, 3000 nanometers) on a side. Inaccordance with some embodiments of the invention, the cross-sectionalsize of the microchannel is in the range from 1-2 micrometers by 1-2micrometers. In accordance with some embodiments of the invention, thecross-sectional dimensions of the microchannel can be in a range fromsufficiently small relative to the smallest dimensions of the bacteriato be detected to enable the bacteria to either just pass through themicrochannel or become jammed into the microchannel to sufficientlylarge such that the presence of live bacteria cause detectible changesin the resistance through the microchannel

In operation, a current source I can be used to provide aconstant-amplitude electric current that is injected into reservoir 1and flows through the nanochannel 120 to the electrical ground(reservoirs 3 and 4). The voltage drop between reservoirs 2 and 4 ismeasured by a voltage measuring device V (e.g., an oscilloscope, adigital signal processor, an analog signal processor—a voltmeter, alock-in amplifier, or a computer). Alternatively, a constant amplitudevoltage source may be connected across the microchannel (reservoirs 1and 3). Here, the current flowing through the microchannel may bedetected using a current measuring device (e.g., an ammeter, amultimeter, a lock-in amplifier, a current amplifier, or a computer). Inaccordance with some embodiments of the invention, the contactresistances in the system do not alter the measured signals. Theelectrical impedance of the microchannel 120 is dominated by itsresistance at the low-frequencies used and the effects of the parasiticcapacitances are thus negligible.

FIGS. 2A-2E show diagrammatic views of a microchannel 120 according tosome embodiments of the invention. The microscopically small channel 120can be filled with an ionic aqueous solution, such as phosphate-bufferedsaline (PBS), urine, or other biological fluid that can sustain thebacteria to be tested. The effective channel cross-section is typicallyon the order of several micrometers (e.g., 1-2 micrometers by 1-2micrometers) in diameter or smaller (e.g., a rectangular cross-sectionof 1 micrometer by 1 micrometer; also see FIG. 3B, FIG. 6D, FIG. 6E,FIG. 14 and FIG. 15 for representative dimensions and geometries) and isherein referred to as the microchannel 120. The microchannel length canbe longer (e.g., 50-200 micrometers). The microchannel 120 can beconnected to larger mm-sized reservoirs at one or both ends, interfacingwith standard microfluidic elements.

As shown in FIG. 2A, prior to the introduction of any bacteria in thesolution, the intrinsic electrical noise in the circuit can be monitoredand determined. These intrinsic-noise-based electrical fluctuations canbe used to establish a baseline (e.g., a noise floor) of the measurementof bacterial activity. As shown in FIGS. 2B and 2C, bacterial activity(e.g., bacteria 130 moving (e.g., swimming) into the channel from thereservoirs at one or both ends) changes the effective channel diameterand causes changes to the electrical properties of the microchannel(e.g., the electrical resistance of the microchannel). In other words,the effective cross-sectional area of the nanochannel 120 for conductingions is modulated by the presence (or absence) of bacteria 130 in themicrochannel 120. These electrical resistance fluctuations directlycorrespond to bacterial activity. For example, if the bacteria 130 aredead, these fluctuations cease and effectively return to the intrinsiclevel.

FIG. 2A shows an ionic solution in the nanochannel 120. In an exemplaryembodiment, ions flow through a nanochannel 120 under an imposedelectric potential resulting in a measurable electric current I. Thevoltage drop V may be measured as a function of time, and the electricalresistance is R=V/I. In accordance with some embodiments of theinvention, as shown in FIG. 2B a bacterium 130 enters the microchannel120, the electrical resistance increases due to the reduction in theeffective cross-section of the nanochannel 120 diameter. This results ina voltage increase under constant current. As a bacterium exits themicrochannel 120, the resistance decreases due to increase (e.g.,restoration) of the cross-section of the microchannel. This results involtage fluctuations, even though the time averaged value of the voltagemay be stable. The exemplary voltage fluctuations are shown in FIGS. 2Band 2C with the corresponding fluctuation in bacterium.

FIG. 2D shows an ionic solution in the microchannel 120 according tosome embodiments of the invention, however, all or a portion of thewalls of the microchannel 120 are treated such that the bacteria cells132 become attached, adhered or tethered to the treated walls ofmicrochannel 120. Alternatively, the cross-section of microchannel canbe small such that the cells become physically stuck or jammed in themicrochannel. In this embodiment, the nanomechanical movements (e.g.,wiggling) of the bacteria 132 cause corresponding fluctuations in theelectrical resistance, similar to the situation described above. Theresistance fluctuations arise because the bacterial movements modulatethe electrical resistance through the channel. When the bacteria 132die, the resistance fluctuations decrease due to the decrease in themovements of the bacteria and the modulations of the effectivecross-section of the microchannel 120.

FIG. 2E shows an ionic solution in the microchannel 120 according tosome embodiments of the invention, however, all or a portion of thewalls of the microchannel 120 are treated such that the bacteria cell134 become attached, adhered or tethered to the treated walls ofmicrochannel 120. In accordance with some embodiments of the invention,the bacteria 134 proliferate in the microchannel 120 and new bacteriacells 136 grow in the microchannel and change the mean resistance byconstricting the microchannel 120. The mean or average resistance can bedetermined as a function of the measured resistance through themicrochannel. The measured resistance can be determined (e.g., using ohmlaw) by applying a constant voltage to microchannel and measuring thecurrent over time or applying a constant current to the microchannel andmeasuring the voltage over time.

In some embodiments, the measured electrical signals (e.g., V and R) inthe microchannel 120 are compared before and after administering theantibiotic. Referring now to FIG. 2F, in accordance with someembodiments of the invention, a chip 100 can be designed with severalmicrochannels 120 connected to a reservoir 110. The measurement circuitcan be multiplexed in the device such that it can read out theresistances of each of the microchannels at the same time. The samplecan be delivered/pipetted into reservoir 110 of the chip 100, whichfirst divides the sample using, for example, gravity feed or amicrofluidic pump and channels. In other words, the sample is initiallyaliquoted into the several microchannels. This step can be automatedusing programmable microfluidics. Once the sample is delivered into themicrochannels in parallel, the measurement procedure can be run asdiscussed, but with each microchannel being exposed to a differentantibiotic.

In accordance with some embodiments, a signal processing system can beused to measure the voltage and/or current signal fluctuations. Thesignal fluctuations of the base solution before the bacteria are addedcan be measured and used as a baseline signal to evaluate the signalfluctuations after live bacteria are added. The signal processing systemcan monitor signal fluctuations by measuring the amplitude of the signal(e.g., over the baseline signal) to detect live bacteria in themicrochannel. These higher amplitude signal fluctuations should becomereduced after the introduction of the antibiotic and the signal shouldreturn to the base level. The signal processor can include a computerprocessor and associated memory that stores computer programs that canbe executed by the processor to process the signals and identify thefluctuations and change in amplitude to indicate the presence of livebacteria in the microchannel, as well as the susceptibility of thebacteria to antibiotic treatment by the reduction in the higheramplitude signals.

In accordance with some embodiments of the invention, the microchannel120 can be fabricated using silicone (PDMS) and other biocompatiblematerials using well established processing steps. In accordance withsome embodiments of the invention, the microchannel 120 can bepositioned between two (or more) mm-sized reservoirs with exemplarylinear dimensions of w×h×l≈2 μm×2 μm×100 μm. In alternative embodiments,the linear dimensions may be varied to attain more efficient detectionof the motility and the fluctuations based on the size of the bacteriato be evaluated. When motile bacteria in solution are introduced intothe antibiotic susceptibility testing system 100 via the reservoirs 110at one or both ends, the bacteria swim randomly and some enter themicrochannel 120 region.

FIGS. 2A-2F show how the chip 100 according the various embodiments ofthe invention can be used as a transducer that converts bacterialviability (e.g., motion and growth) into electrical signals usingdifferent modalities. There are negligible electrical fluctuations inthe microchannel without any bacteria (FIG. 2A) and it behaves as anelectrical resistor, which has time-independent resistance. Inaccordance with some embodiments of the invention, planktonic motilebacteria 130 swimming in the microchannel generate fluctuations (FIGS.2B and 2C). In accordance with some embodiments of the invention,bacteria 132 captured/immobilized inside the microchannel also generatesubstantial electrical fluctuations owing to their random movements(wiggling or oscillations), as shown in FIG. 2D. In this embodiment,bacteria 132 can become tethered to the microchannel surface byadhesion-promoting coatings and/or simply adhere to the surface bythemselves and/or become jammed into the tight microchannel. Thus, therandom movements of bacteria 132 modulate the microchannel diameter(FIG. 2D), thereby resulting in detectable electrical signals. Anychanges in bacterial 132 viability and movements are sensitivelycaptured in the electrical signal. In accordance with some embodimentsof the invention, the chip 100 can be used to detect bacterial viabilityfrom proliferation. As shown in FIG. 2E, the microchannel device can beused as an ultrasensitive culture medium. As surface-immobilizedbacteria 134 proliferate (e.g., bacteria cells 136 grow) in themicrochannel, the time-averaged (dc) voltage signal increases underconstant electric current proportionally to the number of cells in themicrochannel. This is because the cells simply obstruct the electriccurrent flow and increase the mean (average) electrical resistance.

More insight can be gained from the circuit diagram (FIG. 1C) and therelated equations. Using the circuit diagram in FIG. 1C, Ohm's Law canbe applied:

V _(dc) +δV(t)=I _(dc) ×[R+δR(t)]  (1)

where I_(dc) is a fixed current and R is the time-averaged (dc)electrical resistance of the microchannel. The instantaneous movementsof the bacteria change the electrical resistance by δR(t). Thus, thefluctuating component of the signal that corresponds to bacterialactivity (e.g., live movements) is δV(t)=I_(dc)×δR(t). Finally, thetime-averaged (dc) signal, V_(dc)=I_(dc) R, is proportional to thenumber of cells inside the microchannel due to the obstruction of thecurrent by the cells. The method thus provides both the movement signaland the number of bacteria in the microchannel. Alternatively, thesystem can apply a dc voltage across the microchannel and measure thecurrent fluctuations due to the changes in the resistance of themicrochannel. The equation given by Ohm's Law then becomes:

I _(dc) +δI(t)=V _(dc) /[R+δR(t)]  (2)

Similar to above, the fluctuating component of the signal thatcorresponds to bacterial activity (e.g., live movements) isδI(t)=V_(dc)×δR(t)/R². As above, the time-averaged signal thatcorresponds to the number of cells inside the microchannel is:I_(dc)=V_(dc)/R. As a person having ordinary skill would understand, theanalyses presented in Equations 1 and 2 are valid for alternatingcurrent (AC) signals as well. In the case of AC signals, the amplitudesof the current and voltage would be included in the equations.

FIG. 3A exemplifies a general detection approach to demonstrate theefficiency of the antibiotic susceptibility testing system in accordancewith some embodiments of the invention. First, the baseline for thedevice and the solution can be established by measuring the signal(e.g., voltage or current fluctuations) for a predefined period (e.g. 30sec. to 5 min or more). The measuring time can depend on the solutionused and the device configuration, and some device and solutioncombinations may require more measurement time. The RMS value of thebase signal fluctuations can be computed as a function of the measuredsignal (e.g., voltage or current) fluctuations for one or morepredefined periods of time (e.g. over the measurement time). This isshown as the black signal (area A) in FIG. 3A. Next, bacteria will beintroduced into the device. After a few minutes (e.g., 1-3 min.) ofsettling time, signal data can be collected for a predefined period(e.g. 2 min. to 10 min.) and RMS value of the signal fluctuation causedby the active bacteria can be computed as a function of the collectedsignal data for one or more predefined periods of time. Depending on theconcentration and the character of the bacteria, the settling time andsignal data collection time can be longer or shorter. This is shown asthe red signal (area B) in FIG. 3A. The RMS magnitude of the blacksignal in area A (e.g, before the bacteria were added) and the RMSmagnitude of the red signal in area B (e.g., after the bacteria wereadded) can be compared to determine the signature of the live bacteria.Depending on the nature of the bacteria, the RMS signal magnitude of thered signal (area B) is expected to be approximately 1.2 to 50 timeslarger than the base amplitude (e.g., the black signal in area A). Seealso, FIGS. 11, 12, and 13. The ratio of the active bacteria signal(e.g., red signal in area B) to base signal (e.g., the black signal inarea A) can provide a signature value for the viability of the bacteriaand can be used to set a threshold for detecting the efficacy of anantibiotic treatment for the bacteria being evaluated. For example,after the baseline and bacteria signals are measured and RMS valuesdetermined, antibiotics can be administered, and the fluctuations can bemeasured over a predetermined amount of time, the green signal (area C)in FIG. 3A. The RMS value as a function of (wait) time can be computedin order to determine a measure of the susceptibility of the bacteria tothe antibiotic treatment. In accordance with some embodiments of theinvention, the antibiotic can be considered effective if the RMS signalamplitude returns to the baseline amplitude (or within 10% of thebaseline amplitude) after a certain time interval. The length of thegreen data trace (e.g., area C) shows that some time is required toevaluate the antibiotic's effect on the bacteria as the antibiotic killsthe bacteria, the signal fluctuation should return to or near thebaseline level. The length of time it takes for the signal fluctuationto return to the base level will vary depending on the antibiotic andits interaction with the bacteria.

As a person having ordinary skill will appreciate, different bacteriaand different antibiotics may produce different RMS magnitudes (e.g.,compared to the data in the figure) and may require longer or shortertime periods to stabilize. For example, some types of non-motilebacteria may produce different signal fluctuation amplitudes and RMSamplitude values. Also different antibiotics can take more or less timeto kill the bacteria and the time can vary depending on environmentalfactors, such as temperature, pH and concentration (of the solution,bacteria, and/or the antibiotic).

FIGS. 3B and 3C show testing performed to demonstrate the efficiency ofthe antibiotic susceptibility testing system 100 according to someembodiments of the invention. FIG. 3B shows approximately 10 E. colibacteria cells swimming in the microchannel 120. FIG. 3C shows thevoltage fluctuations recorded in the microchannel over time. Themicrochannel was prepared and filled with a phosphate-buffered saline(PBS) and the voltage fluctuations were measured for approximately 10minutes. As shown in FIG. 3C, the area labeled PBS shows the initialaverage resistance of the channel filled with PBS (e.g., approximately 3MΩ) and the baseline voltage fluctuations are shown. Next, a solution ofPBS containing live E. coli was then pipetted into the reservoirs onboth ends of the microchannel. The concentration of E. coli wasapproximately 10⁸ cells/ml. This E. coli solution changed the measuredaverage resistance by only 1%; in other words, the resistance was stilldominated by the ions in the solution. However, as shown by the centersection (labeled Bacteria) in FIG. 3C, the magnitude of the voltagefluctuations increased significantly. The signal fluctuations shown inthe center section (labeled Bacteria) of FIG. 3C corresponds to theobserved presence of approximately 10 E. coli cells in the microchannel120 (shown in FIG. 3B labeled with arrows). Finally, a small volume ofantibiotic solution was pipetted into the reservoirs of the device; thisdid not change the overall electrical resistance significantly. However,as shown in FIG. 3C in the area labeled ABX, the antibiotic waseffective against the bacteria causing the fluctuations to return to thebaseline uV voltage fluctuation level after approximately 10 min ofadministering the antibiotic.

The data in FIG. 3C shows that the source of the observed increase inthe voltage fluctuations is bacterial activity. It is noted that theaverage electrical resistance in the device (i.e., the microchannel 120)stays about the same throughout the experiment and is dominated by theionic solution. In these experiments performed with motile bacteria, themicrochannel 120 becomes partially “clogged,” and the ionic current inthe channel is strongly modulated by the bacteria swimming in themicrochannel 120 (FIG. 3C). This is because the cross-section of themicrochannel 120 is comparable to the size of a single cell of E. coli(200 nm×200 nm×2 mm). Bacterial cells remained intact after theadministration of the antibiotic (Kanamycin), but their activitystopped. This is supported by the notion that bacterial activity is thesource of the observed signals: dead bacteria move only due to Brownianmotion, which results in mean-square displacements that are smaller byorders of magnitude. This small Brownian motion is either not registeredin our measurements or can be positioned below the noise floor.

To further evaluate the system, further experiments were conducted anddata using different bacteria concentrations were collected. Themicrochannels and other experimental conditions were kept nominallyidentical (e.g., linear dimensions, concentration of PBS solutions).Regardless, there were slight differences in some of the measuredparameters because of unavoidable statistical deviations in thefabrication process. The standard deviation, σ₀, of the baselinefluctuations shifted slightly from device to device (by roughly 30%).Therefore, each experiment was normalized with its own σ₀. Providedbelow is Table 1, detailing the results of the experiments with E. coli.A total of 14 experiments were performed. Each entry is an average of3-4 experiments. The first column is the concentration of bacteria. Thesecond column R is the average resistance measured during the experimentusing 1 nA of electrical current. The third column is the standarddeviation, σ₀, of the baseline fluctuations (e.g., voltage) without anybacteria in the device. Note that σ₀ shifts slightly from experiment toexperiment. The fourth column is the standard deviation σ_(B) of thefluctuations (e.g., voltage) with bacteria.

TABLE 1 Concentration R σ₀ σ_(B) (CFU/ml) (MΩ) (V) (V) σ_(B)/σ₀ 1.0 ×10⁸ 3.0 5.7 × 10⁻⁶ 13.1 × 10⁻⁶ 2.3 2.3 × 10⁸ 2.8 4.2 × 10⁻⁶ 10.1 × 10⁻⁶2.4   3 × 10⁸ 2.9 4.4 × 10⁻⁶ 12.6 × 10⁻⁶ 2.9

Table 1 displays representative data along with the ratio of thestandard deviation, σ_(B), of the fluctuations with bacteria to σ₀measured in the same device, i.e., σ_(B)/σ₀. The higher bacteriaconcentration in the reservoirs corresponds to stronger fluctuations inthe measured potential through the microchannel. More bacteria appear toenter the microchannel 120 region when a higher bacteria concentrationis present in the reservoir. The relationship between the increase inthe fluctuations and bacteria concentration does not appear to be linearand is probably a function involving other factors.

FIG. 4 shows a workflow for antibiotic susceptibility testing accordingto some embodiments of the invention. In this method, bacteria are firstcaptured/immobilized in the microchannel. Then, their viability isdetected from electrical measurements under administered antibiotics.The system according to the invention can be used to provide twopossible and complementary approaches for bacteria viability assessment(and hence antibiotic susceptibility testing). In themean-resistance-based approach, growth or proliferation is detected(FIG. 2E). In the fluctuation-based approach, viability is determinedfrom movements (FIG. 2D).

FIG. 4 shows a flow chart of a method 400 for antibiotic susceptibilitytesting according to some embodiments of the invention. The method 400can include a Mean Resistance based Susceptibility Test 410 and/or anElectrical Fluctuation based Susceptibility Test 430. The method 400 caninclude, at 302, loading the reservoir 110 of one or more chips (e.g.,chip 101) with an ionic solution and bacteria to be tested. At 404,fluid pressure and/or an electric field can be used to move bacteriainto the microchannel 120, wherein the bacteria become trapped, capturedor immobilized in the microchannel 120. The system can determine thatthe bacteria are trapped, captured or immobilized by monitoring the meanresistance signals (e.g., as bacteria are trapped, the mean resistancevalue increases as explained above in Equation 1). At 406, the systemcan determine the mean resistance value and wait a period of time anddetermine the mean resistance value again and compare the two signals tosee if they are different by more than a predefined threshold amount. Ifthe mean resistance values do not differ by more than the predefinedthreshold amount (e.g., the N or No condition, the bacteria are trappedcondition is not true), the system continues to cause the bacteria toflow into the microchannel and then returns to 406 to determine the meanresistance values another time and compares it to the mean resistancevalues of the previous signal. At a later time, the subsequent meanresistance values will be greater than the previous the mean resistancevalues signal by the predefined threshold amount (and the Y or Yes,bacteria are trapped condition becomes true), and at that point eitherthe Mean Resistance based Susceptibility Test 410 or the ElectricalFluctuation based Susceptibility Test 430, or both can be initiated.

After the bacteria are trapped, for the Mean Resistance basedSusceptibility Test 410, at 412, the baseline mean (average ortime-averaged measured resistance through the channel) resistance can bemeasured for a predefined period of time. The baseline mean can bedetermined as a function (e.g., the average or time-average) of themeasured resistance through the channel for two or more points in time.After the baseline mean resistance is determined, at 414, the antibioticis added to the solution, either via one of the reservoirs 110 ordirectly to the microchannel 120 and, at 416, the solution containingthe bacteria and the antibiotic is allowed to incubate for a predefinedperiod of time (e.g., 1-3 hours, although shorter or longer periods canbe used). After the expiration of a predefined period of time, at 418,the mean resistance is determined (e.g., as a function of the measuredresistance through the channel for two or more points in time) and, at420, the mean resistance is compared to the baseline mean resistance. Ifthe mean resistance is greater than the baseline mean resistance, thenthe bacteria can be considered resistant to the antibiotic at 422. Ifthe mean resistance is not greater than the baseline mean resistance,the bacteria can be considered susceptible to attack by the antibioticat 424.

After the bacteria are trapped, for the Electrical Fluctuation basedSusceptibility Test 430, at 432, the system measures the fluctuations ofthe electrical signals (e.g., voltage or current) to determine abaseline RMS signal level (e.g., the RMS level determined as function ofthe measured signal fluctuations). Next, at 434, the antibiotic is addedto the solution, either via one of the reservoirs 110 or directly to themicrochannel 120. Next, at 436, the (RMS) fluctuations of the electricalsignal (e.g., voltage or current) are measured again and an RMS levelcan be determined as a function of the measured signal fluctuations,and, at 440, the RMS level of the fluctuations of the electrical signalcan be compared to the baseline RMS signal level. If the RMS level ofthe fluctuations of the electrical signal is greater than the baselinesignal RMS level, then the bacteria can be considered resistant to theantibiotic at 442. If the RMS level of the fluctuations of theelectrical signal is not greater than the baseline RMS signal level, thebacteria can be considered susceptible to attack by the antibiotic at444.

In accordance with some embodiments of the invention, the geometry ofthe microfluidic channel can be varied to attain more efficientdetection of the motility and the fluctuations. A variation forantibiotic susceptibility testing is shown in FIGS. 5A, 5B, and 5C. Inprevious studies, antibiotic diffusion from reservoirs takes time. Anobject of the present embodiment is to reduce or eliminate unnecessarylatency. As shown in FIG. 5A, an embodiment demonstrates the addition ofan antibiotic reservoir 510 directly connected to the microchannel 120.Furthermore, the pores 514 in the wall between the reservoir and themicrochannel are large enough that antibiotic molecules can pass intothe microchannel, but small such that bacteria cannot pass into theantibiotic reservoir 510. Note that the resistance measurement as notedbefore is not perturbed in this variation. Once the antibiotic isintroduced into the reservoir it quickly diffuses to the microchannel120 region, and the susceptibility testing can be initiated. Thefabrication of the pores 514 can be achieved by using advancedlithography (e.g., electron beam lithography); alternatively, a porousmaterial can be used as the top wall of the device, instead of PDMS. Theantibiotic (ABX) can reach the bacteria in the microchannel 120 quicklyif pores 514 are made on the microchannel wall 512, connecting it to areservoir 510 of antibiotics.

In addition, high currents (e.g., from 250 nA to 10 mA) can be used todrive bacteria in the solution through the device. In particular, thebacteria can be pushed into the microchannel region (e.g., using thehigh current/voltage signal). This can be useful in testing low bacteriaconcentration solutions—the applied high current can be used to speed upthe bacteria migration into the microchannel region. In accordance withsome embodiments of the invention, an electrical trap can be used totrap the bacteria in the microchannel region for efficient detection.For example, FIG. 5B shows an example of the system 100 configured withelectrical guiding/trapping electrodes formed on a substrate thatsupports the microchannel and the reservoirs of the antibioticsusceptibility testing system. As shown in FIG. 5B, electrodes can bedesigned and fabricated on the chip for controlling and trapping thebacteria in the microchannel region for faster and more efficientmeasurements.

In accordance with some embodiments of the invention as shown in FIG.5C, a solution of non-motile bacteria 138 can be introduced into thedevice from one reservoir 110A. A relatively high voltage V can beapplied to move the non-motile bacteria to the constriction at the leftend of the microchannel. Once the bacteria 138 are accumulated as shown,the high voltage is turned off and a low constant current can beactuated for use in measuring the fluctuations in voltage. In accordancewith some embodiments of the invention, the basic electrical fluctuationmeasurement can be performed as follows: 1) a constant direct currentcan be induced through the microchannel and the ensuing alternativecurrent fluctuations in voltage can be measured and monitored.Alternatively, a constant DC voltage can be applied across themicrochannel and current fluctuations can be measured and monitored.

FIGS. 6A-6D show a process for the capture and/or immobilization ofbacteria in the microchannel 120 according to some embodiments of theinvention. The bacteria solution (e.g., urine) can be pushed through themicrochannel by establishing either a pressure-driven flow or anelectrokinetic flow. Motile bacteria (e.g., E. coli) can be immobilizedby specific and non-specific surface chemistry; or by using a high flowforce causing the bacteria cells to jam into the tight microchannel.Non-motile bacteria settle and stick to the microchannel typically ontheir own. After the capture/immobilization, the bacteria do not leavethe microchannel but continue their nanomechanical movements. Inaccordance with some embodiments of the invention, capturing andimmobilizing the bacteria allows for monitoring the viability of a smallpopulation of cells as shown in FIG. 6C.

FIG. 6D shows E. coli immobilized in a microchannel by flow. Themicrochannel tapers from a width of 30 μm down to 1 μm; the entire taperregion in FIG. 6D has a height of about 1 μm. The third dimension (i.e.,the microchannel height) is useful for the capture and the measurements.The measured electrical signals correspond to bacterial movements in theentire taper region. To increase the capture efficiency, themicrochannel surface can be coated with an adhesion promoting layer,such as Poly-D-Lysine (PDL), which is a positively charged amino acid(FIG. 6D inset). Shortly after the flow was established, cellsaccumulated in the taper and adhered to the surface. During this step,the mean (dc) electrical resistance was monitored and was used todetermine how many bacteria were immobilized in the taper region (i.e.,microscope is not needed). There are about 50 cells in FIG. 6D,including those in the constriction, all of which contribute to theelectrical fluctuations that were measured. A selective layer such as acoating including an antibody can also be applied to the microchannelsurface to capture the bacteria selectively.

An advantage of this approach is that it can increase the effectivelocal density of bacteria in the microchannel region by orders ofmagnitude in a time frame of 10s of minutes. For example, see FIG. 6E,which shows a sample with a concentration of about 10⁵ CFU/ml that waspipetted into the device (FIG. 6A), creating a pressure-driven flow. Theflow carries the non-motile cells (S. epidermidis) into the microchannelregion steadily and deposits them there. The images taken at t=0, 2 min.and 5 min. show that a significant density increase can be achieved in arelatively short time.

As previously stated, motile bacteria generate electrical fluctuationsby swimming into and out of the microchannel region. Similarly, livebacteria adhered to a surface demonstrate similar electricalfluctuations and can be measured and detected using electrical signalsin a microchannel or microchannel in accordance with the principals ofthe present invention. Thus, present invention can be used to detect andmonitor bacteria bound to a substrate as well as planktonic bacteria ina channel. FIG. 7A shows a bacterium adhered onto a surface exhibitingnanomechanical fluctuations. As shown in FIG. 7B, the chemical orphysical bond between the bacterium and the surface can be modeled as aspring wherein the bacterium exerts a steady but random force on thespring, resulting in large amplitude (10s of nm) random vibrations.

FIGS. 8A and 8B show a system according to some embodiments of theinvention wherein non-motile bacteria can be detected and monitored totest for antibiotic susceptibility. In accordance with some embodimentsof the invention, non-motile bacteria (e.g., S. epidermidis) can beadhered onto the surfaces of a microchannel. The nanomechanicalfluctuations of the bacteria on the surface will randomly modulate themicrochannel diameter, thereby resulting in detectable electrical signalfluctuations. In other words, the nanomechanical fluctuations of thebacteria adhered on the channel walls will produce the electrical(voltage) fluctuations indicative of the presence of live bacteria inthe channel. Upon administering antibiotics, the bacterial activity willstop and the electrical fluctuations will subside.

FIG. 8A shows an electrostatic interaction can be formed between thechannel wall, and E. coli to adhere the bacteria on the microchannelwalls. The microchannel acquires a negative surface charge whenhydrated. In contrast, the bacteria have negatively charged cell walls.Therefore, by subsequently coating the channel surfaces withPoly-D-Lysine (PDL), which is a positively charged amino acid, byflowing a PDL solution through the microchannel, the bacteria can becaused to adhere easily to the PDL coating via electrostatic forces.FIG. 8B shows the nanomechanical models of the microchannel walls withadhered bacteria, exemplifying the springs model the PDL and areelectrostatic in nature. This process can be used to adhere differenttypes of bacteria onto the microchannel walls. The electrical signalfluctuation measurement can be used to detect whether the bacteria aredead or alive.

FIG. 9A shows a system according to some embodiments of the invention,where correlation measurement can be implemented. In accordance withsome embodiments of the invention, a second pair B of electrodes (V_(B))can be inserted but through separate paths as shown in FIG. 9A. Thecurrent can be applied as before through one electrode pair. Then, thevoltage (V_(A)) fluctuations measured through electrode pair A can becross-correlated with those measured through B: <V_(A)(t)V_(B)(t)>. Inthis embodiment, a more sensitive measurement of the fluctuations can beobtained. Furthermore, the fluctuations not coming from the microchannelregion (i.e., the bacteria) can be reduced and/or eliminated.

FIG. 9B shows a system according to some embodiments of the invention,where radiofrequency (RF) signal techniques can be implemented. Inaccordance with some embodiments of the invention, a low-noise RFamplifier can be used obtain more precise signal measurements. Inaddition, the RF measurements can provide much larger bandwidths and/ormuch smaller time resolution (e.g., latency). Furthermore, in theseembodiments, applying a direct current through the microchannel may notbe necessary. As shown in FIG. 9B, a basic RF reflectometry measurementcircuit is provided wherein RF signal waves are reflected from theelectrodes (701, 702) across the microchannel. Electrode 701 can begrounded and the other electrode 702 can be connected to thereflectometer. This measurement provides the complex impedance, Z(t), ofthe microchannel as a function of time. The imaginary part of theimpedance may change more strongly than the real part (i.e.,resistance), making this measurement useful for bacteria as well asother particles. In accordance with some embodiments of the invention,the system can include the RF transmission measurement shown in FIG. 9C.In both embodiments, an impedance matching circuit can be used (i.e., anLC tank circuit) to better couple the power to the microchannel regionof the circuit.

FIG. 10 shows an antibiotic susceptibility testing system according tosome embodiments of the invention that is adapted to accommodate verysmall sample volumes. In accordance with some embodiments of theinvention, the bacteria can be confined to the microchannel by usingmicrofabricated structures on the channel walls. In accordance with someembodiments of the invention, the relevant sample volume can beapproximately the volume of the microchannel, which is be preconfiguredto accommodate very small sample volumes. FIG. 10 shows an antibioticsusceptibility testing system, which uses an extremely small samplevolume. Here, the bacteria are only introduced into the microchannelregions of the system through a separate inlet. Microfabricated meshesor pillars can be used to separate the microchannel region from themm-sized reservoirs. These meshes can be used restrict bacteria to themicrochannel regions. The ions in the solution can move through themeshes without being hindered. Because the signal (i.e., fluctuations ofbacteria) is coupled to the motion of electrical ions and not the bulkfluid flow, the sensitivity in the device is not degraded. In accordancewith some embodiments of the invention, testing system using thesemicrofabricated meshes and/or pillars can be used to test very smallbacteria volumes (e.g., tens of bacteria). In accordance with someembodiments of the invention, non-motile bacteria can also be tested byadhering the non-motile bacteria to the microchannel surfaces usingfavorable surface chemistry. These embodiments can be used to test smallsample volumes on the order of 10-100 picoliters (10⁻⁸-10⁻⁷ ml).

In accordance with some embodiments of the invention, after capture, theviability of bacteria can be detected from their movements. FIG. 11shows a measured electrical signal (red trace) from about 50 E. colialong with the background (black trace). These cells are shown in theinset of FIG. 11. Once the bacteria cells are jammed or adhered to themicrochannel surface, they do not leave the microchannel region, butcontinue their random nanomechanical oscillations in place that giverise to the observed electrical fluctuations. The root-mean square (RMS)values with and without bacteria are 1.68 μV and 6.31 μV, respectively.

Non-motile bacteria on a surface move incessantly by going throughsubtle random oscillations (wiggles). These movements were firstdetected by means of a microcantilever, see Nanomechanical motion ofEscherichia coli adhered to a surface, C Lissandrello, F Inci, MFrancom, M R Paul, U Demirci, K L Ekinci, Applied physics letters 105(11), 113701, which is hereby incorporated by reference in its entirety.

The data in FIG. 12A shows that these subtle movements of non-motilebacteria are detectable using a nanochannel or microchannel transducerin accordance with the invention. Experiments were performed with S.epidermidis, which is a non-motile bacteria very similar to S. aureusbut less virulent. The cells were adhered to the microchannel walls fromsolution as above in FIGS. 6A-6E. The microscope image in the inset ofFIG. 12B shows that the cells completely fill the tapered microchanneland top half of the tight constriction. The electrolyte filling thedevice is a nutrient broth. FIG. 12A shows two electrical voltage signaltraces as a function of time—the background signal (black) and thefluctuation signal with S. epidermidis in the microchannel (red). Aslight increase can be observed in the fluctuations. A frequency-domainanalysis provides more insight and makes the subtle features in the dataprominent. FIG. 12B shows the power spectra of the fluctuations,obtained numerically from the time-domain signals in FIG. 12A. Thefigures show that the fluctuations have increased significantly atfrequencies in the range 0.3 Hz<f<10 Hz.

Example

FIGS. 13A, 13B, and 13C show the results of antibiotic susceptibilitystudies in human urine. In this example, two strains of E. coli areused, with one being resistant to the administered antibiotic, nalidixicacid. The two strains are immobilized in two separate but identicalchips, such as shown in FIG. 6D. The electrolyte filling the chips is aurine/broth/antibiotic mixture, with the antibiotic concentration setabove the minimum inhibitory concentration (MIC) level. The voltagefluctuations of about 50 bacteria are shown in FIGS. 13A and 13B, as afunction of time, after the antibiotic is administered. The x-axes inFIGS. 13A and 13B show real time. The voltage fluctuations of theresistant strain do not show significant changes over time, asdetermined from the root-mean square (RMS) amplitude of the voltagefluctuations. The voltage fluctuations for the susceptible strain areseen to decay rather quickly. FIG. 13C shows the RMS values of thefluctuations in a bar graph as a function of time for both the resistantand the susceptible strains. This measurement suggests that, inaccordance with some embodiments of the invention, the time for aconclusive test should be approximately 2 hours.

For these experiments, commercially-acquired urine (BioreclamationIVT,Long Island, N.Y.) with the following properties was used: pH=6;resistivity≈10 mS/cm (excellent conductor); with some large crystals(>10 μm). After spiking with bacteria and mixing with broth/antibiotics,the urine was filtered in a large-pore-size filter. The filtered urinewas then directly pipetted into the device as the test matrix. The finalbacteria concentration was about 10⁵-10⁶ CFU/ml, close to clinicalconcentrations in UTI.

In the experiment shown in FIGS. 14, 15 and 16, the susceptibility of S.epidermidis to Vancomycin was tested using the mean resistance basedapproach shown in the flowchart in FIG. 4. As discussed above, S.epidermidis is non-motile and readily settles in the microchannel. Twoidentical chips were prepared, one filled with broth solution as shownin FIG. 14 (and represented by the blue data bars on the right in FIG.16) and the other with broth/Vancomycin mixture as shown in FIG. 15 (andrepresented by the green data bars on the left in FIG. 16). The changesin the mean resistances, which reflect the sizes of the S. epidermidispopulations, are shown as a function of time in the bar graphs of FIG.16. As discussed above, the population in broth continues to grow untilthe microchannel is completely filled. On the other hand, in theantibiotic solution, the population size stays the same (microscopeimages not shown). This is accurately reflected in the mean resistancevalue, which does not change noticeably. In accordance with someembodiments of the invention, the antibiotic susceptibility can bedetermined in a time scale comparable to the cell division time.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Although any known methods, devices, and materials may be used in thepractice or testing of the invention, the methods, devices, andmaterials in this regard are described herein.

Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meaning provided below. Unless explicitly statedotherwise, or apparent from context, the terms and phrases below do notexclude the meaning that the term or phrase has acquired in the art towhich it pertains. The definitions are provided to aid in describingparticular embodiments, and are not intended to limit the claimedinvention. Further, unless otherwise required by context, singular termsshall include plural and plural terms shall include the singular.

As used herein, the term “comprising” or “comprises” is used inreference to compositions, methods, and respective component(s) thereof,that are useful to an embodiment, yet open to the inclusion ofunspecified elements, whether useful or not.

As used herein, the term “consisting essentially of” refers to thoseelements for a given embodiment. The term permits the presence ofelements that do not materially affect the basic and novel or functionalcharacteristic(s) of that embodiment of the invention.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±1% of the value being referred to. For example, about 100 meansfrom 99 to 101.

The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

What is claimed is:
 1. A method for antibiotic susceptibility testingcomprising providing a constant current or constant voltage through amicrochannel wherein the microchannel does not contain an bacteria andmeasuring baseline voltage or current fluctuations through themicrochannel to determine a baseline voltage or current fluctuationsignal; providing a constant current or constant voltage through amicrochannel wherein the microchannel contains at least one bacteriumand measuring voltage or current fluctuations through the microchannelto determine a first voltage or current fluctuation signal; anddetermining that the at least one bacterium is alive if the firstvoltage or current fluctuation signal is greater than the baselinevoltage or current fluctuation signal by a predefined threshold amount.2. The method according to claim 1 wherein the baseline voltage orcurrent fluctuation signal is determined as a function of the measuredbaseline voltage or current fluctuations and the first voltage orcurrent fluctuation signal is determined as a function of the measuredvoltage or current fluctuations.
 3. The method according to claim 2wherein the baseline voltage or current fluctuation signal includes anRMS signal level determined as a function of the measured baselinevoltage or current fluctuations and the first voltage or currentfluctuation signal includes an RMS signal level determined as a functionof the measured voltage or current fluctuations.
 4. A system forantibiotic susceptibility testing comprising: a microchannel extendingfrom a first end to second end; at least one reservoir connected to themicrochannel; a voltage source or a current source connected to thefirst end and the second end of the microchannel to produce asubstantially constant electric potential or a substantially constantelectric current through the microchannel; and a signal measuring devicefor measuring changes in the current or voltage through the microchannelover time.
 5. The system for antibiotic susceptibility testing of claim4, wherein bacteria in the microchannel modulate the resistance orconductance through the microchannel and the signal measuring devicemeasures the modulated current or voltage through the microchannel as anindication of live bacterial activity in the microchannel.
 6. The systemfor antibiotic susceptibility testing of claim 5, wherein the signalmeasuring device detects a baseline signal when no bacterial activityoccurs in the microchannel.
 7. A method for antibiotic susceptibilitytesting comprising: flowing bacteria into a microchannel whereby thebacteria become trapped in the microchannel; measuring a firstresistance through the microchannel after the bacteria become trapped inthe microchannel and determining a first mean resistance through themicrochannel; adding an antibiotic to the microchannel; waiting apredefined period of time; measuring a second resistance through themicrochannel after the bacteria become trapped in the microchannel anddetermining a second mean resistance through the microchannel; comparingthe first mean resistance to the second mean resistance; and determiningthat the bacteria are resistant to the antibiotic if the second meanresistance is greater than the first mean resistance.
 8. The method forantibiotic susceptibility testing according to claim 7 wherein the firstmean resistance is determined as a function of the measured firstresistance and the second mean resistance is determined as function ofthe measured second resistance.
 9. The method for antibioticsusceptibility testing according to claim 8 wherein the measured firstresistance includes two or more first resistance measurements and thefirst mean resistance is determined as a function of at least two of thetwo or more first resistance measurements, and measured secondresistance includes two or more second resistance measurements and thesecond mean resistance is determined as function of at least two of thetwo or more second resistance measurements.
 10. A method for antibioticsusceptibility testing comprising: flowing bacteria into a microchannelwhereby the bacteria become trapped in the microchannel; measuringvoltage or current signal fluctuations through the microchannel afterthe bacteria become trapped in the microchannel and determining a firstmeasure of the voltage or current signal fluctuations through themicrochannel; adding an antibiotic to the microchannel; measuringvoltage or current signal fluctuations through the microchannel afterthe bacteria become trapped in the microchannel and determining a secondmeasure of the voltage or current signal fluctuations through themicrochannel; comparing the first measure of voltage or current signalfluctuations to the second measure of voltage or current signalfluctuations; and determining that the bacteria are resistant to theantibiotic if the second measure of voltage or current signalfluctuations is greater than the first measure of voltage or currentsignal fluctuations.
 11. The method for antibiotic susceptibilitytesting according to claim 10 wherein first voltage signal fluctuationsare measured and the first measure of voltage signal fluctuations isdetermined as a function of the Root Mean Square (RMS) level of themeasured first voltage signal fluctuations, and second voltage signalfluctuations are measured and the second measure of voltage signalfluctuations is determined as a function of the Root Mean Square (RMS)level of the measured second voltage signal fluctuations
 12. The methodfor antibiotic susceptibility testing according to claim 10 whereinfirst current signal fluctuations are measured and the first measure ofcurrent signal fluctuations is determined as a function of the Root MeanSquare (RMS) level of the measured first current signal fluctuations,and second current signal fluctuations are measured and the secondmeasure of current signal fluctuations is determined as a function ofthe Root Mean Square (RMS) level of the measured second current signalfluctuations